Diseases affecting the lung epithelium are not easily treatable and result in significant morbidity and mortality worldwide. Specialized stem cells with the potential to self-renew or give rise to differentiated, functional progeny have been proposed as a critical component of tissue homeostasis for many organs, including the lung. Different potential approaches for the use of stem cells for lung disease treatment include enhancement of endogenous stem cell differentiation or in vitro directed differentiation of stem cells to lung lineages followed by cell transplantation. Both approaches require that the identity and pathways of differentiation of lung stem or progenitor cells be known and well characterized. Embryonic stem cells (ESCs) have emerged in the last 10- 15 years as a promising platform for the development of cell-based therapies, since they can transit through several defined stages in vitro to recapitulate mammalian development. In addition, induced pluripotent stem cells (iPSCs) offer an attractive alternative to human ESCs. iPSCs are easy to derive, are not fraught with ethical issues and offer the possibility of patient-specific therapies. Although most protocol use soluble factors to derive the desired cell types it is gradually recognized that cell-matrix interactions and stiffness of the cell culture substratum have important roles in directed differentiation of ESCs and iPSCs. Thus, the overall objective of this project is to undertake the first systematic study of the role of biomechanical cues in lung specification and differentiation n an in vitro system of lung development. This represents the first step towards our long-term goal of developing cell-based therapies for diseases affecting the lung epithelium. In Aim 1 we will develop an essential tool which is a combinatorial platform of extracellular matrix (ECM) proteins on gels of various stiffness. We will use this platform in Aim 2 to identify the optimal combination of ECM proteins and substratum stiffness for the derivation of lung progenitors and their differentiation. To monitor the emergence of lung progenitors and their differentiated progeny we will use the mouse Nkx2-1-GFP ESC and SPC-GFP iPSC reporter lines, respectively. The optimal biomechanical conditions defined from Aim 2 experiments will be used to derive clinically-relevant lung progenitors from human iPSCs in Aim 3 experiments. These progenitors will then be seeded on decellularized lung scaffolds from normal and fibrotic donors to study the effect of increased lung stiffness on both lung progenitor differentiation and phenotype of differentiated progeny. We envision that the outcome of our studies will be to define the optimal protocol for derivation of lung progenitor cells from ESCs/iPSCs to be used in novel lung disease therapies and to understand how lung stiffness affects the properties of engrafted in vitro derived epithelial lung progenitors.